Reducing optical interference in a fluidic device

ABSTRACT

This invention is in the field of medical devices. Specifically, the present invention provides portable medical devices that allow real-time detection of analytes from a biological fluid. The methods and devices are particularly useful for providing point-of-care testing for a variety of medical applications. In particular, the medical device reduces interference with an optical signal which is indicative of the presence of an analyte in a bodily sample.

CROSS-REFERENCE

This application is a continuation application of Ser. No. 11/549,558,filed Oct. 13, 2006, which is incorporated herein by reference in itsentirety and is related to the co-pending patent application Ser. No.11/389,409, filed Mar. 24, 2006, now U.S. Pat. No. 7,635,594 which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The discovery of a vast number of disease biomarkers and theestablishment of miniaturized fluidic systems have opened up new avenuesto devise methods and systems for the prediction, diagnosis andmonitoring of treatment of diseases in a point-of-care setting.Point-of-care testing is particularly desirable because it rapidlydelivers results to patients and medical practitioners and enablesfaster consultation between patients and health care providers. Earlydiagnosis allows a practitioner to begin treatment sooner and thusavoiding unattended deterioration of a patient's condition. Frequentmonitoring of appropriate parameters such as biomarker levels andconcentrations of therapeutic agents enables recognition of theeffectiveness of drug therapy or early awareness that the patient isbeing harmed by the therapy. Examples of point-of-care analyses includetests for glucose, prothrombin time, drugs of abuse, serum cholesterol,pregnancy, and ovulation.

Fluidic devices can utilize a number of different assays to detect ananalyte of interest in a sample of bodily fluid from a subject. In ELISAassays (a preferred technique for clinical assays especially in apoint-of care context, if assay reagents such as enzyme-antibodyconjugates and enzyme substrates remain on-board the fluidic deviceafter the assay is performed, reagents unbound to the assay capturesurface or excess reagents, if collected in the same fluidic device, canreact with one another and create a signal that can interfere with thesignal of interest produced by the assay. This is especially the case inluminogenic assays in which the assay reagents generate light, incontrast to assays that measure, for example, absorbance orfluorescence. Many luminogenic assays use an enzyme to generateluminescence thus improving assay sensitivity by amplification of themeasured species. Moreover, in assay systems that contain all assaycomponents, including waste washes in a small housing the potential forglowing luminogenic waste materials is further enhanced. In such assayformats, the excess or unbound enzyme-labeled reagent may react withenzyme substrate, thus creating undesired interfering signals.

Some fluidic device features may mitigate the problem of an interferingsignal to a certain degree. For example, the body of the fluidic devicecan be opaque, optically isolating the undesired glow, or the detectionsystem can be configured to reject light which does not originate fromreaction sites within the fluidic device. These mitigating features,however, may not sufficiently eliminate the interference as light canstill travel through transparent elements of the fluidic device andinterfere with the signal of interest. This is especially the case inassays requiring high sensitivity where the ratio between the signalgenerated from the assay may represent only a small fraction, e.g., lessthan 1 part in 10,000, of the total signal generating reagent.

Thus, there remains a considerable need for improved fluidic devices,especially point-of-care devices, designed to minimize interferingoptical signals.

SUMMARY OF THE INVENTION

One aspect of the invention is a fluidic device for detecting an analytein a sample of bodily fluid. The fluidic device comprises a samplecollection unit adapted to provide a sample of bodily fluid into thefluidic device, an assay assembly in fluidic communication with thesample collection unit, wherein the assay assembly is adapted to yieldan optical signal indicative of the presence or quantity of the analytein the sample of bodily fluid, and a quencher assembly in fluidiccommunication with said assay assembly, wherein the quencher assembly isadapted to reduce interference of the optical signal.

In some embodiments the assay assembly includes reagent chamberscomprising reagents used in the assay and at least one reaction sitecomprising a reactant that binds the analyte. The reagents can be anenzyme conjugate and an enzyme substrate.

The quencher assembly can include quenching site in fluidiccommunication with the reaction site and a quenching agent at thequenching site. The quencher assembly can also include an absorbentmaterial, which may be, for example, glass fiber, silica, paper,polyacylamide gel, agarose, or agar.

The absorbent material can be impregnated with the quenching agent. Thequenching agent can be adapted to inactivate at least one reagent fromthe assay and thereby reduce the interfering optical signal. In someembodiments the quenching agent is4-amino-1,11-azobenzene-3,41-disulfonic acid.

In some embodiments the assay assembly is adapted to run an immunoassay,which can be a chemiluminescent assay. The quencher assembly can beadapted to substantially eliminate the interference.

In some embodiments the fluid device has a waste chamber, wherein thewaste chamber includes the quenching site.

Another aspect of the invention is a system for detecting an analyte ina sample. The system comprises a fluidic device that has an assayassembly configured to yield an optical signal that is indicative of thepresence of the analyte, and a quencher assembly in fluidiccommunication with said assay assembly, wherein said quencher assemblyis adapted to reduce interference of said optical signal, and adetection assembly for detecting said optical signal.

In some embodiments the system also includes a communication assemblyfor transmitting said optical signal to an external device.

In some embodiments the assay assembly comprises reagent chambers thathave at least one reagent used in the assay and at least one reactionsite comprising a reactant that binds the analyte. The at least onereagent can include an enzyme conjugate and an enzyme substrate.

In some embodiments the quencher assembly comprises a quenching site influidic communication with the reaction site and a quenching agent atthe quenching site. The quencher assembly can include an absorbentmaterial such as glass fiber, silica, paper, polyacylamide gel, agarose,or agar. The absorbent material can be impregnated with the quenchingagent, which be adapted to inactivate at least one reagent from saidassay, thereby reducing said interference of said optical signal. Thequenching agent can be, for example,4-amino-1,11-azobenzene-3,41-disulfonic acid.

In some embodiments of the system, the assay assembly is adapted to runan immunoassay, and can further be a chemiluminescent assay.

The quencher assembly can be adapted to substantially eliminate theinterference.

In some embodiments of the system there is a waste chamber, wherein thewaste chamber comprises the quenching site.

One aspect of the invention is a method of detecting an analyte in asample. The method comprises allowing a sample suspected to contain theanalyte to react with reagents contained in a fluidic device that has anassay assembly configured to yield an optical signal that is indicativeof the presence of the analyte, and a quencher assembly in fluidiccommunication with said assay assembly, wherein said quencher assemblyis adapted to reduce interference of said optical signal, and detectingsaid optical signal thereby detecting the analyte in the sample.

One aspect of the invention is a method of manufacturing a fluidicdevice having a quencher assembly. The method includes providing aplurality of layers of the fluidic device, affixing said layers togetherto provide for a fluidic network between a sample collection unit, atleast one reagent chamber, at least one reaction site, and at least onequencher assembly.

In some embodiments the affixing comprising ultrasonic welding thelayers together.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1 and 2 show top and bottom views of an exemplary fluidic device,illustrating the fluid connectivity.

FIGS. 3 and 4 show a top and bottom view, respectively, of an exemplaryfluidic of the present invention.

FIG. 5 illustrates the different components and layers of an exemplaryfluidic device.

FIG. 6 shows an exemplary system of the present invention.

FIG. 7 shows a two-step assay.

FIG. 8 depicts an exemplary chemiluminescent assay.

DETAILED DESCRIPTION OF THE INVENTION Fluidic Device

One aspect of the invention is a fluidic device for detecting an analytein a sample of bodily fluid. The fluidic device includes an assayassembly configured to yield an optical signal that is indicative of thepresence of the analyte in the sample and an quencher assembly adaptedto reduce interference of the optical signal.

The fluidic device generally has a sample collection unit, an assayassembly, fluidic channels and one or more waste chambers.

The sample collection unit comprises an inlet for receiving a sample,e.g., a bodily fluid from a subject, such as blood or urine, optionallya receptacle for holding the sample, and an outlet to a fluidic channelthat connects with the assay assembly.

The assay assembly comprises (a) at least one and optionally a pluralityof reagent chambers, optionally containing reagents to perform adetection assay, (b) at least one and, optionally, a plurality ofreaction sites, each site comprising a surface to which is immobilized areactant that recognizes and binds an analyte, and (3) fluidic channelsthat connect the reaction sites with the sample collection unit and thereagent chambers. The assay provides an optical signal indicative of thepresence of an analyte of interest, which can then be detected by adetection assembly as described below. Unbound, or excess, sample andreagents remain on-board the fluidic device after the assay, and collectin a waste chamber within the fluidic device.

A reactant immobilized at a reaction site can be anything useful fordetecting an analyte of interest in a sample of bodily fluid. Forinstance, such reactants include without limitation nucleic acid probes,antibodies, cell membrane receptors, monoclonal antibodies andpolyclonal antibodies reactive with a specific analyte. Variouscommercially available reactants such as a host of polyclonal andmonoclonal antibodies specifically developed for specific analytes canbe used.

In some embodiments there are more than one reaction sites which canallow for detection of multiple analytes of interest from the samesample of bodily fluid. In some embodiments there are 2, 3, 4, 5, 6, ormore reaction sites, or any other number of reaction sites as may benecessary to carry out the present invention.

In embodiments with multiple reaction sites on a fluidic device, eachreaction site may be immobilized with a reactant different from areactant on a different reaction site. In a fluidic device with, forexample, three reaction sites, there may be three different reactants,each bound to a different reaction site to bind to three differentanalytes of interest in the sample. In some embodiments there may bedifferent reactants bound to a single reaction site if, for example, aCCD with multiple detection areas were used as the detection device,such that multiple different analytes could be detected in a singlereaction site. Exemplary reaction sites are more fully described inpatent application Ser. No. 11/389,409, filed Mar. 24, 2006, which isincorporated by reference herein in its entirety.

Assay detection relies on luminescence and, in particular,chemiluminescence. In one embodiment, the assay employs an enzymeconjugate comprising, e.g., a protein conjugated with an enzyme. Theenzyme can react with a substrate to generate a luminescent signal. Itis contemplated that the assay can be a direct assay, a competitiveassay, in which a reactant not bound to an analyte is exposed to areagent comprising an analyte molecule conjugated to the enzyme, or asdescribed below for detecting small molecules, the two-step assay.Another preferred assay that can be performed using the subject deviceis an Enzyme-Linked ImmunoSorbent Assay (“ELISA”). In anotherembodiment, a fluorescent dye is coupled to or used in tandem with achemiluminescent reaction to further amplify the signal to obtain awider range of linear response.

The assay assembly preferably comprises all of the reagents necessary toperform the assay in the fluidic device. Such reagents are on-board, orhoused within the fluidic device before, during, and after the assay. Inthis way the only inlet for liquids from the fluidic device ispreferably the bodily fluid sample initially provided to the fluidicdevice. There is preferably no liquid outlet in the fluidic device,however does enter and leave the fluidic device during the assay as ameans of propelling liquid movement. An advantage is using such anon-board system is the easily disposable nature of the fluidic device,as well as attempting to prevent leakage from the fluidic device onto orinto a detection device used to detect an optical signal produced duringthe assay which is indicative of the presence of an analyte of interestin the sample. Furthermore all potentially biohazardous materials arecontained within the cartridge.

The reagent chambers within the assay assembly are preferably in fluidiccommunication with at least one reaction site, and when the fluidicdevice is actuated to initiate the flow of fluid as described herein,reagents contained in the reagent chambers are released into the fluidicchannels.

Reagents according to the present invention include without limitationwash buffers, enzyme substrates, dilution buffers, conjugates,enzyme-labeled conjugates, DNA amplifiers, sample diluents, washsolutions, sample pre-treatment reagents including additives such asdetergents, polymers, chelating agents, albumin-binding reagents, enzymeinhibitors, enzymes, anticoagulants, red-cell agglutinating agents,antibodies, or other materials necessary to run an assay on a fluidicdevice. An enzyme conjugate can be a conjugate with a polyclonalantibody, monoclonal antibody, hapten or other member of a binding pair(MBP) label that can yield a detectable signal upon reaction with anappropriate enzyme substrate. Non-limiting examples of such enzymes arealkaline phosphatase and horseradish peroxidase. In some embodiments thereagents comprise immunoassay reagents.

Reagent chambers can contain approximately about 50 μl to about 1 ml offluid. In some embodiments the chamber may contain about 100 μl offluid. The volume of liquid in a reagent chamber may vary depending onthe type of assay being run or the sample of bodily fluid provided.

Liquids and other substances described herein that are transportedthrough the fluidic device flow through channels within the fluidicdevice. Such channels will typically have small cross sectionaldimensions. In some embodiments the dimensions are from about 0.01 mm toabout 5 mm, preferably from about 0.03 mm to about 3 mm, and morepreferably from about 0.05 mm to about 2 mm. Fluidic channels in thefluidic device may be created by, for example without limitation, dyecutting, machining, precision injection molding, laser etching, or anyother techniques known in the art.

The fluidic device also preferably includes a quencher assembly. Thequencher assembly comprises a quenching site in fluid communication withthe reaction site and a quenching agent at the quenching site. Thequenching assembly can optionally comprise an absorbent materialimpregnated with the quenching agent. In certain embodiments, thequenching site comprises or is part of the waste chamber.

The quencher assembly is typically adapted to reduce an optical signalfrom the fluidic device that interferes with the optical signalindicative of the presence of the analyte in the sample. In someembodiments the quencher assembly reduces the interfering optical signalby at least about 50%, at least about, 60%, at least about 70%, at leastabout 80%, at least about 90%, or more. In preferred embodiments thequencher assembly reduces optical interference by at least about 99%. Inanother preferred embodiment the quenching assembly reduces interferenceby at least about 99.5%. In more preferred embodiments the quencherassembly reduces optical interference by about 99.9%.

In some embodiments the quencher assembly can be adapted to physicallyblock optical interference from the waste liquid. For example, thequencher assembly can comprise a dark coating to absorb opticalinterference from the fluidic device.

In some embodiments the quencher assembly can comprise an absorbentmaterial impregnated with the quenching agent. In general, an absorbentmaterial is a material or substance that has the power or capacity ortendency to absorb or take up liquid. Absorption mechanisms can includecapillary forces, osmotic forces, solvent or chemical action, or othersimilar mechanisms.

An absorbent material can be a solid material, porous material, gel,porous or sintered polymer, high salt fluid, thermoplastic polymer (suchas those available from Sigma, Aldrich, Porex™, etc.), polyolefin resin,or porous plastic, including, e.g., Porex™ plastics. The absorbentmaterial may also be an aerosol particulate spray, for example,comprising porous particulate matter.

The absorbent material can be a cellulosic material such as paper, e.g.,Kimwipe™, paper towel or the like.

The absorbent material can also be, for example, polyacrylamide,agarose, agar, polyethylene, polypropylene, a high molecular weightpolyethylene, polyvinylidene fluoride, ethylene-vinyl acetate,polytetrafluoroethylene, stryene-acrylonitrile, polysulfone,polycarbonate, dextran, dry sephadex, polyhthalate, silica, glass fiber,or other material similar to those included herein. Additionally, anabsorbent material can be any combination of the materials describedherein.

In general the absorbent material is bibulous and the volume fraction ofair is generally about 10-70% of the absorbent material. The absorbentmaterial helps absorb waste liquids used in the assay and thereforeprevents leakage of fluid from the fluidic device, as may be desirableto prevent contamination on or into a detection device used inconjunction with the fluidic device to detect the optical signal.

In some embodiments the absorbent material comprises at least onequenching agent which reacts with at least one reagent from said assayassembly to reduce interference of the optical signal indicative of thepresence of the analyte in the sample. The quenching agent can inhibitthe binding between reagents, or in preferred embodiments the quenchingagent inactivates at least one and more preferably all reagents whichmay contribute to an interfering optical signal.

The reagent or reagents with which the quenching agent in the quencherassembly reacts to reduce the interference can be, for example withoutlimitation, an unbound enzyme and/or an unbound substrate. The reagentwith which the quenching agent reacts to reduce the interference isgenerally not as important as the reduction of the interference itself.The quenching agent in the quencher assembly can vary depending on thetype of assay that is being performed in the fluidic device. Preferablya subject quenching agent reduces an interfering optical signal by atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, or more. In a preferred embodiment thequenching agent reduces an interfering optical signal by about 99%. Inanother preferred embodiments the quenching assembly reduces opticalinterference by at least about 99.5%. In more preferred embodiments thequenching agent reduces optical interference by at least about 99.9%.

In this way the quencher assembly can be produced with a specific assayor assays in mind and can comprise quenching agents which willsatisfactorily reduce the interfering signal.

In some embodiments the quenching agent can be a chemical that is astrong non-volatile acid such as trichloroacetic acid or its salt sodiumtrichloracetate. The substance can also be a strong alkali such assodium hydroxide. Other strong non-volatile acids and strong alkalis canbe used in accordance with the present invention.

In some embodiments the quenching agent reduces the optical interferenceby inhibiting the enzyme. In an ELISA, e.g., the quenching agent caninterfere with the enzyme's ability to convert the substrate to producea luminescent signal. Exemplary enzyme inhibitors include lactose whichinhibits the action of β-galactosidase on luminogenic galactosides, andphosphate salts which inhibit phosphatases.

In some embodiments the quenching agent can reduce the interference bydenaturing the enzyme. By denaturing the enzyme it is unable to carryout it enzymatic function and the optical interference is suppressed orreduced. Exemplary denaturants include detergents such as sodium dodecylsulfate (SDS), heavy metal salts such as mercuric acetate, or chelatingagents such as EDTA which can sequester metal ions essential foractivity of certain enzymes such as alkaline phosphatase. All types ofsurfactants may be used including cationic (CTMAB) and anionic (SDS).

In some embodiments the quenching agent can be a non-denaturing chemicalthat is incompatible with enzyme activity. Exemplary chemicals includebuffers and the like that change the pH to a value where the enzymebecomes inactive and thus unable to catalyze the production of theinterfering signal.

In other embodiments the quenching agent can be, for example, an organiccharge-transfer molecule, including 7,7,8,8-tetracyanoquinodimethane(TCNQ), 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane (TFTCNQ),carbon nanotubes, mordant yellow 10 (MY) and4-amino-1,1-azobenzene-3,4-disulfonic acid (AB). In preferredembodiments the azobenzene compounds are MY and AB, as they areconsiderably more water-soluble than TCNQ, TFTCNQ and carbon nanotubes.The structure of AB is shown below in:

In some embodiments the quenching agent can be heavy atoms such asiodine which reduces the interference by quenching a fluorescent speciesused to enhance a chemiluminescent signal. In other embodiments thequenching agent can be an organic compound with an absorption spectrumoverlapping the fluorescence emission spectrum of a fluorescent speciesused to enhance a chemiluminescent signal. In some embodiments such aquenching agent is a dark quencher such as a dispersion of carbonparticles (e.g., carbon black, charcoal). Carbon can inactivatechemiluminescence by absorbing actives species, and it is also a verygood quenching agent that is substantially incapable of emittingfluorescence.

In some embodiments the quenching agent can be an antioxidant, which canreduce the interference by disrupting the chemiluminescent reaction.Quenching agents that may be used in some embodiments of the inventioninclude but are not limited to Trolox, butylated hydroxytoluene (BHT),ascorbic acid, citric acid, retinol, carotenoid terpenoids,non-carotenoid terpenoids, phenolic acids and their esters, andbioflavinoids.

In other embodiments, the quenching agent can be a singlet oxygenquencher, which can reduce the interference by disrupting thechemiluminescent reaction. Some singlet oxygen quenchers include but arenot limited to 1,4 diazabicyclo [2,2,2]octane, thiol containingcompounds such as methionine or cysteine, and carotenoids such aslycopene.

The substance used to impregnate or saturate the absorbent material ispreferably highly concentrated, typically in large molar excess of theassay reagents.

Generally the quencher assembly possesses desirable certain propertiessome of which, by way of example, are now described. In embodiments inwhich the quencher assembly comprises an absorbent material, theabsorption of waste liquids is preferably fast relative to the durationof the assay. In preferred embodiments the absorption of waste liquidsoccurs within a few minutes and more preferably within a few seconds.

The absorbent material preferably absorbs substantially all of theliquid waste in the fluidic device. In preferred embodiments more than99% of the liquid in the waste chamber is absorbed. In addition toreducing the optical interference, this helps prevent liquid fromleaking from the fluidic device after the assay is complete, which helpsprevent contamination of a detection device as may be used with thefluidic device as described herein.

The quencher assembly's inhibition of enzyme activity should preferablybe rapid, typically within a few minutes and more preferably within afew seconds.

The inhibitory enzyme reaction should be as complete as possible toensure the interference is reduced as much as possible. In preferredembodiments the inactivation of the enzyme reaction should be more than99% complete before the optical signal indicative of the presence of theanalyte in the sample is detected by any detection mechanism that may beused with the fluidic device as described herein.

In preferred embodiments the quencher assembly comprises an absorbentmaterial, and as such, the inactivating substance imbedded therein ispreferably stable within the absorbent material. Furthermore, thequenching agent preferably dissolves within seconds to minutes of beingexposed to the waste liquids.

In some embodiments the quencher assembly comprises the waste chamber. Awaste chamber is generally a chamber or well in fluidic communicationwith the assay assembly in which assay reagents and sample which do notbind to the reaction site in the assay assembly collect after the assay.As the waste fluids remain on-board the fluidic device after the assay,the waste chamber is generally the area of the fluidic device in whichany unbound or excess reagents and sample collect after the assay. Inembodiments in which the quencher assembly comprises an absorbentmaterial, the absorbent material may be adapted to be housed within thewaste chamber. The absorbent material may or may not fill up the entirewaste chamber, and may expand when a fluid enters the waste chamber.

The quencher assembly may also comprise a stabilizing feature adapted tostabilize or secure the absorbent material within the fluidic device.For example, a waste chamber adapted to house the absorbent material mayalso comprise a pin or stake projecting from the top of the wastechamber to contact and stabilize or secure the absorbent pad.

FIGS. 1 and 2 show a top and bottom view, respectively, of an exemplaryfluidic device after the device has been assembled. The different layersare designed and affixed to form a three dimensional fluidic channelnetwork. A sample collection unit 4 provides a sample of bodily fluidfrom a patient. As will be explained in further detail below a readerassembly comprises actuating elements (not shown) can actuate thefluidic device to start and direct the flow of a bodily fluid sample andassay reagents in the fluidic device. In some embodiments actuatingelements first cause the flow of sample in the fluidic device 2 fromsample collection unit 4 to reaction sites 6, move the sample upward inthe fluidic device from point G′ to point G, and then to waste chamber 8in which absorbent material 9 is housed. The actuating elements theninitiate the flow of reagents from reagent chambers 10 to point B′,point C′, and point D′, then upward to points B, C, and D, respectively,then to point A, down to point A′, and then to waste chamber 8 in thesame manner as the sample. When the sample and the reagents enter thewaste chamber 8 they encounter quencher assembly 9.

To ensure that a given photon count produced at a reaction sitecorrelates with an accurate concentration of an analyte of interest in asample, it is preferably advantageous to calibrate the fluidic devicebefore detecting the photons. Calibrating a fluidic device at the pointof manufacturing for example may be insufficient to ensure an accurateanalyte concentration is determined because a fluidic device may beshipped prior to use and may undergo changes in temperature, forexample, so that a calibration performed at manufacturing does not takeinto effect any subsequent changes to the structure of the fluidicdevice or reagents contained therein. In a preferred embodiment of thepresent invention, a fluidic device has a calibration assembly thatmimics the assay assembly in components and design except that a sampleis not introduced into the calibration assembly. Referring to FIGS. 1and 2, a calibration assembly occupies about half of the fluidic device2 and includes reagent chambers 32, reactions sites 34, a waste chamber36, fluidic channels 38, and absorbent material 9. Similar to the assayassembly, the number of reagent chambers and reaction sites may varydepending on the assay being run on the fluidic device and the number ofanalytes being detected.

FIG. 3 is a top view of another exemplary embodiment of a fluidicdevice. A plurality of absorbent materials 9 are shown. FIG. 4 shows abottom view of the embodiment from FIG. 3.

FIG. 5 illustrates the plurality of layers of the exemplary fluidicdevice shown in FIGS. 3 and 4. The position of absorbent material 9 isshown relative to the other components and layers of the fluidic device.

A detection assembly as shown in FIG. 6 then detects the optical signalindicative of the presence of the analyte in the sample, and thedetected signal can then be used to determine the concentration of theanalyte in the sample. FIG. 6 illustrates the position of an exemplarydetection assembly that can be used to detect an optical signal from thefluidic device that is indicative of the presence of an analyte ofinterest in the sample. The detection assembly may be above or below thefluidic device or at a different orientation in relation to the fluidicdevice based on, for example, the type of assay being performed and thedetection mechanism being employed.

In preferred embodiments an optical detector is used as the detectiondevice. Non-limiting examples include a photomultiplier tube (PMT),photodiode, photon counting detector, or charge-coupled device (CCD).Some assays may generate luminescence as described herein. In someembodiments chemiluminescence is detected. In some embodiments adetection assembly could include a plurality of fiber optic cablesconnected as a bundle to a CCD detector or to a PMT array. The fiberoptic bundle could be constructed of discrete fibers or of many smallfibers fused together to form a solid bundle. Such solid bundles arecommercially available and easily interfaced to CCD detectors.

Exemplary detection assemblies that may be used with the fluidic deviceare described in patent application Ser. No. 11/389,409, filed Mar. 24,2006, which is incorporated by reference herein in its entirety.

Interference, or optical interference, as described herein generallymeans an optical signal produced in the fluidic device which interfereswith the optical signal produced by bound reactants, which is indicativeof the presence of an analyte of interest. Typically, such aninterfering signal is produced in the waste chamber where the reagentswhich do not bind to the reaction sites accumulate and encounter oneanother. The accumulation of waste liquids can produce such interferencewhen, for example, an enzyme used in an assay to increase assaysensitivity reacts with an unbound substrate, creating an optical signalthat interferes with the optical signal generated by bound reactants.

Method of Use

Another aspect of the invention is a method of detecting an analyte in asample. The method comprises allowing a bodily fluid sample suspected tocontain the analyte to react with reactants contained in a fluidicdevice which has an assay assembly configured to yield an optical signalthat is indicative of the presence of the analyte and a quencherassembly adapted to reduce interference of said optical signal, anddetecting the optical signal thereby detecting the analyte in thesample.

Any sample of bodily fluids suspected to contain an analyte of interestcan be used in conjunction with the subject system or devices. Commonlyemployed bodily fluids include but are not limited to blood, serum,saliva, urine, gastric and digestive fluid, tears, stool, semen, vaginalfluid, interstitial fluids derived from tumorous tissue, andcerebrospinal fluid. In some embodiments, the bodily fluids are provideddirectly to the fluidic device without further processing. In someembodiments, however, the bodily fluids can be pre-treated beforeperforming the analysis with the subject fluidic devices. The choice ofpre-treatments will depend on the type of bodily fluid used and/or thenature of the analyte under investigation. For instance, where theanalyte is present at low level in a sample of bodily fluid, the samplecan be concentrated via any conventional means to enrich the analyte.Where the analyte is a nucleic acid, it can be extracted using variouslytic enzymes or chemical solutions according to the procedures setforth in Sambrook et al. (“Molecular Cloning: A Laboratory Manual”), orusing nucleic acid binding resins following the accompanyinginstructions provided by manufactures. Where the analyte is a moleculepresent on or within a cell, extraction can be performed using lysingagents including but not limited to denaturing detergent such as SDS ornon-denaturing detergent such as Thesit®, sodium deoxylate, tritonX-100, and tween-20.

A bodily fluid may be drawn from a patient and brought into the fluidicdevice in a variety of ways, including but not limited to, lancing,injection, or pipetting. In some embodiments, a lancet punctures theskin and draws the sample into the fluidic device using, for example,gravity, capillary action, aspiration, or vacuum force. In anotherembodiment where no active mechanism is required, a patient can simplyprovide a bodily fluid to the fluidic device, as for example, couldoccur with a blood or saliva sample. The collected fluid can be placedin the sample collection unit within the fluidic device where thefluidic device can automatically detect the required volume of sample tobe used in the assay. In yet another embodiment, the fluidic devicecomprises at least one microneedle which punctures the skin. Themicroneedle can be used with a fluidic device alone, or can puncture theskin after the fluidic device is inserted into a reader assembly. Samplecollections techniques which may be used herein are described in patentapplication Ser. No. 11/389,409, filed Mar. 24, 2006, which isincorporated by reference herein in its entirety.

In some embodiments a sample of bodily fluid can first be provided tothe fluidic device by any of the methods described herein. The fluidicdevice can then be inserted into a reader assembly as shown in FIG. 6.An identification detector housed within the reader assembly can detectan identifier of the fluidic device and communicate the identifier to acommunication assembly, which is preferably housed within the readerassembly. The communication assembly then transmits the identifier to anexternal device which transmits a protocol to run on the fluidic devicebased on the identifier to the communication assembly. A controllerpreferably housed within the reader assembly controls actuating elementsincluding at least one pump and one valve which interact with thefluidic device to control and direct fluid movement within the device.The reader assembly and its components illustrated in FIG. 6 are morefully described in patent application Ser. No. 11/389,409, filed Mar.24, 2006, which is incorporated by reference herein in its entirety.

The fluidic device is preferably initially calibrated using acalibration assembly by running the same reagents as will be used in theassay through the calibration reaction sites, and then an optical signalfrom the reactions sites is detected by the detection means, and thesignal is used in calibrating the fluidic device. Calibration techniquesthat may be used in the fluidic device herein can be found in patentapplication Ser. No. 11/389,409, filed Mar. 24, 2006, which isincorporated by reference herein in its entirety. The sample containingan analyte is introduced into the fluidic channel. The sample may bediluted, mixed, and/or and further separated into plasma or otherdesired component using a filter. The sample then flows through thereaction sites and analytes present therein will bind to reactants boundthereon. The sample fluid is then flushed out of the reaction wells intoa waste chamber. Depending on the assay being run, appropriate reagentsare directed through the reaction sites via the channels to carry outthe assay. Any wash buffers and other reagents used in the varioussteps, including the calibration step, are collected in at least onewaste chamber. The signal produced in the reaction sites is thendetected by any of the detection methods described herein.

A variety of assays may be performed in a fluidic device according tothe present invention to detect an analyte of interest in a sample.

The detection assay relies on luminescence and, in particular,chemiluminescence. In one embodiment, the assay employs an enzymeconjugate comprising, e.g., a protein conjugated with an enzyme. Theenzyme can react with a substrate to generate a luminescent signal. Itis contemplated that the assay can be a direct assay or a competitiveassay, in which a reactant not bound to an analyte is exposed to areagent comprising an analyte molecule conjugated to the enzyme.Further, a fluorescent compound may be used in tandem or coupled withthe chemiluminescent reaction, in order to linearly multiply the signaloutput of the reaction.

In an exemplary two-step assay shown in FIG. 7, the sample containinganalyte (“Ag”) first flows over a reaction site containing antibodies(“Ab”). The antibodies bind the analyte present in the sample. After thesample passes over the surface, a solution with analyte conjugated to amarker (“labeled Ag”) at a high concentration is passed over thesurface. The conjugate saturates any of the antibodies that have not yetbound the analyte. Before equilibrium is reached and any displacement ofpre-bound unlabelled analyte occurs, the high-concentration conjugatesolution is washed off. The amount of conjugate bound to the surface isthen measured by the appropriate technique, and the detected conjugateis inversely proportional to the amount of analyte present in thesample.

An exemplary measuring technique for a two-step assay is achemiluminescence enzyme immunoassay as shown in FIG. 8. As is known inthe field, the marker can be a commercially available marker such as adioxetane-phosphate, which is not luminescent but becomes luminescentafter hydrolysis by, for example, alkaline phosphatase. An enzyme suchas alkaline phosphatase is exposed to the conjugate to cause thesubstrate to luminesce. In some embodiments the substrate solution issupplemented with enhancing agents such as, without limitation,fluorescein in mixed micelles, soluble polymers, or PVC which create amuch brighter signal than the luminophore alone. The mechanism by whichthe quencher assembly functions to reduce interference is not criticalto the functionality of the present invention, as long as theinterference is reduced by a sufficient amount.

An ELISA is another exemplary assay for which an optical quencher can beused to remove an interfering signal generated by reactants to areaction site. In a typical ELISA, a sample containing an antigen ofinterest is passed over the reaction site, to which analytes of interestin the sample will bind by virtue of antibody molecules (directed to theantigen) adsorbed to the reaction site. Then, enzyme-labeled antibodyconjugate (directed to the antigen and selected such that the antibodybound to the reaction site does not block binding of the conjugate) ispassed over the reaction site, allowed to bind, then displaced by thesubstrate. Enzyme causes the substrate to produce an optical signal.Unbound reagents which end up in the waste chamber can similarly produceinterfering signals.

In some embodiments the label is coupled directly or indirectly to amolecule to be detected such as a product, substrate, or enzyme,according to methods well known in the art. As indicated above, a widevariety of labels are used, with the choice of label depending on thesensitivity required, ease of conjugation of the compound, stabilityrequirements, available instrumentation, and disposal provisions. Nonradioactive labels are often attached by indirect means. Generally, aligand molecule is covalently bound to a polymer. The ligand then bindsto an anti-ligand molecule which is either inherently detectable orcovalently bound to a signal system, such as a detectable enzyme, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with labeled,anti-ligands. Alternatively, any haptenic or antigenic compound can beused in combination with an antibody.

In some embodiments the label can also be conjugated directly to signalgenerating compounds, for example, by conjugation with an enzyme.Enzymes of interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidoreductases,particularly peroxidases. Chemiluminescent compounds include luciferin,and 2,3-dihydrophthalazinediones, such as luminol, dioxetanes andacridinium esters.

Methods of detecting labels are well known to those of skill in the art.Detection can be accomplished using of electronic detectors such asdigital cameras, charge coupled devices (CCDs) or photomultipliers andphototubes, or other detection devices. Similarly, enzymatic labels aredetected by providing appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally, simple colorimetriclabels are often detected simply by observing the color associated withthe label. For example, conjugated gold often appears pink, whilevarious conjugated beads appear the color of the bead.

Suitable chemiluminescent sources include a compound which becomeselectronically excited by a chemical reaction and may then emit lightwhich serves as the detectable signal. A diverse number of families ofcompounds have been found to provide chemiluminescence under a varietyor conditions. One family of compounds is2,3-dihydro-1,4-phthalazinedione. A frequently used compound is luminol,which is a 5-amino compound. Other members of the family include the5-amino-6,7,8-trimethoxy- and the dimethylamino[ca]benz analog. Thesecompounds can be made to luminesce with alkaline hydrogen peroxide orcalcium hypochlorite and base. Another family of compounds is the2,4,5-triphenylimidazoles, with lophine as the common name for theparent product. Chemiluminescent analogs include para-dimethylamino and-methoxy substituents. Chemiluminescence may also be obtained withoxalates, usually oxalyl active esters, for example, p-nitrophenyl and aperoxide such as hydrogen peroxide, under basic conditions. Other usefulchemiluminescent compounds that are also known include —N-alkylacridinum esters and dioxetanes. Alternatively, luciferins may be usedin conjunction with luciferase or lucigenins to provide bioluminescence.

In some embodiments immunoassays are run on the fluidic device. Whilecompetitive binding assays, which are well known in the art, may be runin some embodiments, in some embodiments a two-step method is used whicheliminates the need to mix a conjugate and a sample before exposing themixture to an antibody, which may be desirable when very small volumesof sample and conjugate are used, as in the fluidic device of thepresent invention. A two-step assay has additional advantages over thecompetitive binding assays when use with a fluidic device as describedherein. It combines the ease of use and high sensitivity of a sandwich(competitive binding) immunoassay with the ability to assay smallmolecules.

An exemplary two-step assay shown in FIG. 8 has been described herein,as has an exemplary measuring technique for the two-step assay—achemiluminescence enzyme immunoassay as shown in FIG. 8.

The term “analytes” according to the present invention includes withoutlimitation drugs, prodrugs, pharmaceutical agents, drug metabolites,biomarkers such as expressed proteins and cell markers, antibodies,serum proteins, cholesterol, polysaccharides, nulceic acids, biologicalanalytes, biomarkers, genes, protein, or hormones, or any combinationthereof. At a molecular level, the analytes can be polypeptide,proteins, glycoprotein, polysaccharide, lipid, nucleic acid, andcombinations thereof.

A more complete list of analytes which can be detected using a fluidicdevice and methods described herein are included in patent applicationSer. No. 11/389,409, filed Mar. 24, 2006, which is incorporated byreference herein in its entirety.

One aspect of the invention is a method of manufacturing a fluidicdevice having a quencher assembly. The method comprises providing aplurality of layers of the fluidic device, and affixing the layers toprovide for a fluidic network between a sample collection unit, at leastone reagent chamber, at least one reaction site, and at least one wastechamber comprising an quencher assembly.

In some embodiments at least one of the different layers of the fluidicdevice may be constructed of polymeric substrates. Non limiting examplesof polymeric materials include polystyrene, polycarbonate,polypropylene, polydimethysiloxanes (PDMS), polyurethane,polyvinylchloride (PVC), polymethylmethacrylate and polysulfone.

Manufacturing of the fluidic channels may generally be carried out byany number of microfabrication techniques that are well known in theart. For example, lithographic techniques are optionally employed infabricating, for example, glass, quartz or silicon substrates, usingmethods well known in the semiconductor manufacturing industries such asphotolithographic etching, plasma etching or wet chemical etching.Alternatively, micromachining methods such as laser drilling,micromilling and the like are optionally employed. Similarly, forpolymeric substrates, well known manufacturing techniques may also beused. These techniques include injection molding. Stamp molding andembossing methods where large numbers of substrates are optionallyproduced using, for example, rolling stamps to produce large sheets ofmicroscale substrates or polymer microcasting techniques where thesubstrate is polymerized within a micromachined mold. Dye casting mayalso be used.

In preferred embodiments the different layers of the fluidic device areultrasonically welded together according to methods known in the art.The layers may also be coupled together using other methods, includingwithout limitation, stamping, thermal bonding, adhesives or, in the caseof certain substrates, e.g., glass, or semi-rigid and non-rigidpolymeric substrates, a natural adhesion between the two components.

FIG. 5 shows an embodiment of the invention in which a fluidic device 2is comprised of a plurality of different layers of material. Features asshown are, for example, cut in the polymeric substrate such that whenthe layers are properly positioned when assembly will form a fluidicnetwork. In some embodiments more or fewer layers may be used toconstruct a fluidic device to carry out the purpose of the invention.

The quencher assembly has been described herein and in some embodimentscan comprise an absorbent material. In such embodiments the quencherassembly can be produced by applying the quenching agent into theabsorbent material. This can be accomplished by any number of techniqueswell known in the art, such as pipetting the liquid onto the absorbentmaterial until the absorbent material is substantially imbedded in theabsorbent material, or simply allowing the absorbent material to absorbthe quenching agent. The amount of saturation of the absorbent materialmay vary, as long as a sufficient amount of the quenching agent isincorporated into the absorbent material to produce an inhibitory effecton at least assay reagent.

After the quenching agent is added to the absorbent material, theabsorbent material is then dried. The drying step can be accomplished byany suitable technique such as freeze drying, drying under flowing gaswith temperature elevation, or simply passive drying by allowing waterin the absorbent material to evaporate.

Once dry, the absorbent material incorporating the quenching agent canthen be placed into a fluidic device as described during themanufacturing process where it can be used to reduce opticalinterference in an assay performed within the fluidic device. Theplacement inside the fluidic device can be by any known technique andcan simply be manually placing it into the fluidic device. As describedabove, the absorbent material is preferably placed in a waste chamberadapted to collect unbound liquids used inside the fluidic device.

Example

A 1×0.5 inch piece of Whatman #32 glass fiber mat (item 10 372 968) wasimpregnated with 50 uL of 15% w/v 4-amino-1,1-azobenzene-3,4-disulfonicacid (0.4 M) in water then dried in a “dry box”.

In assays using alkaline-phosphatase (from bovine intestine)-labeledreagents (APase coupled to haptens or to antibodies at concentrations ofup to about 10 ug/mL in a dilute tris buffer) and either Lumigen'sLumiphos™ 530, or KPL Phosphoglow™ AP substrates (both are dioxetanesand have an esterified phosphate residue on which the enzyme acts) usedas supplied by the vendors (100 uM in dioxetane), the result was about200 uL of enzyme and 200 uL of substrate in the waste chamber, thusexposed to the adsorbent material.

After an initial glow rate of 38,550 counts/second (observed by placingthe fluidic device in a Molecular Devices M5 luminometer such that thewaste chamber was being interrogated), the intensity dropped to about100 counts/second within a few seconds after adding the adsorbentmaterial (the noise level of the luminometer was about 100counts/second). In other words, more than 99% of the opticalinterference was eliminated.

The azobenzene acted in an inhibitory manner on both the enzyme and thesubstrate. The enzyme was inactivated by the acidity of the reagent, andlikely by other mechanisms as well. The substrate was chemicallymodified by the azobenzene such that it is no longer a substrate foralkaline phosphatase.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A fluidic device for detecting an analyte in a sample of bodilyfluid, comprising: a sample collection unit adapted to provide a sampleof bodily fluid into said fluidic device; an assay assembly in fluidiccommunication with said sample collection unit, wherein said assayassembly is adapted to yield an optical signal indicative of thepresence of the analyte in the sample of bodily fluid; and a quencherassembly in fluidic communication with said assay assembly, wherein saidquencher assembly is adapted to reduce interference of said opticalsignal.
 2. The fluidic device of claim 1, wherein said assay assemblycomprises reagent chambers comprising at least one reagent used in saidassay and at least one reaction site comprising a reactant that bindssaid analyte.
 3. The fluidic device of claim 2, wherein said at leastone reagent comprises an enzyme conjugate and an enzyme substrate. 4.The fluidic device of claim 2, wherein said quencher assembly comprisesa quenching site in fluidic communication with said reaction site and aquenching agent at said quenching site.
 5. The fluidic device of claim4, wherein said quencher assembly further comprises an absorbentmaterial.
 6. The fluidic device of claim 5, wherein said absorbentmaterial is impregnated with said quenching agent.
 7. The fluidic deviceof claim 5, wherein said absorbent material is selected from the groupconsisting of glass fiber, silica, paper, polyacylamide gel, agarose,and agar.
 8. The fluidic device of claim 5, wherein said quenching agentis adapted to inactivate at least one reagent from said assay, therebyreducing said interference of said optical signal.
 9. The fluidic deviceof claim 8, wherein said quenching agent is4-amino-1,11-azobenzene-3,41-disulfonic acid.
 10. The fluidic device ofclaim 1, wherein said assay assembly is adapted to run achemiluminescent assay.
 11. The fluidic device of claim 1, wherein saidquencher assembly is adapted to substantially eliminate saidinterference.
 12. The fluidic device of claim 1, wherein said fluidicdevice is adapted to run an immunoassay.
 13. The fluidic device of claim4, further comprising a waste chamber, wherein said waste chambercomprises said quenching site.
 14. The fluidic device of claim 1,wherein said sample of bodily fluid is blood.
 15. A system for detectingan analyte in a sample, comprising: a fluidic device comprising an assayassembly configured to yield an optical signal that is indicative of thepresence of the analyte; and a quencher assembly in fluidiccommunication with said assay assembly, wherein said quencher assemblyis adapted to reduce interference of said optical signal; and adetection assembly for detecting said optical signal.
 16. The system ofclaim 15, further comprising a communication assembly adapted totransmit said optical signal to an external device.
 17. The system ofclaim 15, wherein said assay assembly comprises reagent chamberscomprising at least one reagent used in said assay and at least onereaction site comprising a reactant that binds said analyte.
 18. Thesystem of claim 17, wherein said at least one reagent comprises anenzyme conjugate and an enzyme substrate.
 19. The system of claim 17,wherein said quencher assembly comprises a quenching site in fluidiccommunication with said reaction site and a quenching agent at saidquenching site.
 20. The system of claim 19, wherein said quencherassembly further comprises an absorbent material.
 21. The system ofclaim 20, wherein said absorbent material is impregnated with saidquenching agent.
 22. The system of claim 20, wherein said absorbentmaterial is selected from the group consisting of glass fiber, silica,paper, polyacylamide gel, agarose, and agar.
 23. The system of claim 20,wherein said quenching agent is adapted to inactivate at least onereagent from said assay, thereby reducing said interference of saidoptical signal.
 24. The system of claim 23, wherein said quenching agentis 4-amino-1,11-azobenzene-3,41-disulfonic acid.
 25. The system of claim17, wherein said assay assembly is adapted to run a chemiluminescentassay.
 26. The system of claim 17, wherein said quencher assembly isadapted to substantially eliminate said interference.
 27. The system ofclaim 17, wherein said fluidic device is adapted to run an immunoassay.28. The system of claim 19, further comprising a waste chamber, whereinsaid waste chamber comprises said quenching site.
 29. The system ofclaim 15, wherein said sample of bodily fluid is blood.
 30. A method ofdetecting an analyte in a sample, comprising: allowing a samplesuspected to contain the analyte to react with at least one reactantcontained in a fluidic device that comprises: an assay assemblyconfigured to yield an optical signal that is indicative of the presenceof the analyte; and an quencher assembly in fluidic communication withsaid assay assembly, wherein said quencher assembly is adapted to reduceinterference of said optical signal; and detecting said optical signalthereby detecting the analyte in the sample.
 31. The method of claim 30,wherein said assay assembly comprises reagent chambers comprising atleast one reagent used in said assay and at least one reaction sitecomprising a reactant that binds said analyte.
 32. The method of claim31, wherein said at least one reagent comprises an enzyme conjugate andan enzyme substrate.
 33. The method of claim 31, wherein said quencherassembly comprises a quenching site in fluidic communication with saidreaction site and a quenching agent at said quenching site.
 34. Themethod of claim 33, wherein said quencher assembly further comprises anabsorbent material.
 35. The method of claim 34, wherein said absorbentmaterial is impregnated with said quenching agent.
 36. The method ofclaim 34, wherein said absorbent material is selected from the groupconsisting of glass fiber, silica, paper, polyacylamide gel, agarose,and agar.
 37. The method of claim 34, wherein said quenching agent isadapted to inactivate at least one reagent from said assay, therebyreducing said interference of said optical signal.
 38. A method ofmanufacturing a fluidic device having a quencher assembly, comprising:providing a plurality of layers of the fluidic device; affixing saidlayers together to provide for a fluidic network between a samplecollection unit, at least one reagent chamber, at least one reactionsite, and at least one quencher assembly.
 39. The method of claim 38,wherein said affixing comprising ultrasonically welding said layerstogether.
 40. The method of claim 38, wherein said quencher assemblycomprises a quenching site in fluidic communication with said at leastone reaction site and a quenching agent at said quenching site.
 41. Themethod of claim 40, wherein said quencher assembly further comprises anabsorbent material.
 42. The method of claim 41, wherein said absorbentmaterial is impregnated with said quenching agent.
 43. The method ofclaim 41, wherein said absorbent material is selected from the groupconsisting of glass fiber, silica, paper, polyacylamide gel, agarose,and agar.
 44. The method of claim 42, wherein said quenching agent is4-amino-1,11-azobenzene-3,41-disulfonic acid.
 45. A fluidic device fordetecting an analyte in a sample, comprising: a sample collection unit;an assay assembly comprising at least one reaction site, said reactionsite comprising a surface and a reactant that binds an analyteimmobilized thereto; a first reagent chamber comprising an enzymeconjugate; a second reagent chamber comprising an enzyme substrate,wherein the enzyme substrate reacts with the enzyme conjugate to producea luminescent signal; and fluidic channels that connect the reagentchambers with the at least one reaction site; a quencher assemblycomprising an absorbent material and a quenching agent, wherein thequenching agent diminishes the reaction between the enzyme conjugate andthe enzyme substrate; and a plurality of fluidic channels that connectthe sample collection unit and the quencher assembly with the assayassembly.
 46. The fluidic device of claim 45, wherein said quencherassembly further comprises a waste chamber connected through a fluidchannel with the assay assembly, wherein the waste chamber comprises theabsorbent material.
 47. The fluidic device of claim 45, wherein saidabsorbent material is selected from the group consisting of glass fiber,silica, paper, polyacylamide gel, agarose, or agar.
 48. The fluidicdevice of claim 47, wherein said quenching agent is4-amino-1,11-azobenzene-3,41-disulfonic acid and said enzyme conjugateis an alkaline phosphotase-labeled reagent.
 49. The fluidic device ofclaim 45, wherein said assay assembly is adapted to run an immunoassay.